Lipid Association-induced N- and C-terminal Domain Reorganization in Human Apolipoprotein E3

Abstract

Apolipoprotein E (apoE) is a 299 amino acid, antiatherogenic protein that plays a key role in regulating plasma lipoprotein metabolism. It is composed of an N-terminal (NT) domain (residues 1-191) that is responsible for binding to members of the low density lipoprotein receptor family and a C-terminal (CT) domain (residues 216 -299) that anchors the protein to lipoprotein particles by virtue of its high-affinity lipid binding characteristics. Isoform-specific differences in the NT domain that modulate the lipoprotein binding preference elicited by the CT domain suggest the existence and importance of domain interactions in this protein. Employing steady state fluorescence quenching and resonance energy transfer techniques, spatial proximity relationships between the N-and Cterminal domains were investigated in recombinant human apoE3. ApoE3 containing a single Trp at position 264 and an N-iodoacetyl-N-(5-sulfo-1-napthyl) ethylenediamine (AEDANS) moiety covalently attached to the lone Cys residue at position 112 was used (AE-DANS-apoE3/W@264). Fluorescence quenching studies revealed a solvent-exposed location for Trp-264. In the lipid-free state, fluorescence resonance energy transfer (FRET) was noted between Trp-264 and AEDANS, with a calculated distance of 27 Å between the two fluorophores. Control experiments established that FRET observed in this system is intramolecular. FRET was abolished upon proteolysis in the linker region connecting the NT and CT domains. Lowering the solution pH to 4 induced an increase in the efficiency of intramolecular energy transfer, with the two domains reorienting about 5 Å closer to one another. Interdomain FRET was retained in the presence of 0.6 -1.0 M guanidine hydrochloride but was lost at higher concentrations, a manifestation of unfolding of the domains and increased distance between the donoracceptor pair. Interaction of AEDANS-apoE3/W@264 with lipid induced a loss of FRET, attributed to spatial repositioning of the domains by >80 Å. The data provide biophysical evidence that, in addition to reported conformational changes in the four-helix bundle configuration induced by lipid association, lipid binding of apoE is accompanied by reorientation of the tertiary disposition of the NT and CT domains.

Effect of Carboxyl-Terminal Truncation on Structure and Lipid Interaction of Human Apolipoprotein E4

“…This is because the former reflects the number of Trp residues located in the C-terminal domain whereas the latter reflects the number of amino acid residues in this domain. residues were mutated to Phe, leaving a single Trp at position 264 (apoE4 W@264) as a probe for the C-terminal domain (44). This mutation caused no change in structure and stability of the protein (data not shown).…”

The LDL receptor binding domain of apolipoprotein E directs the relative orientation of its C-terminal segment in reconstituted nascent HDL

“…7 . This model takes into consideration the number of apoE molecules that was estimated to be 4–6 per discoidal rHDL based on the following experimental observations and theoretical restrictions, and the assumption that lipid-bound apoE exists in an extended helical conformation: molecular mass and Stokes’ diameter of rHDL ~500 kDa and ~16 nm, respectively from native PAGE (data not shown), discoidal particle geometry from electron microscopy imaging (data not shown), lipid: protein molar ratio of 100:1 from compositional analysis, α-helical conformation as noted previously [ 33 , 46 , 47 ], estimation of POPC bilayer thickness to be ~50 Å [ 48 ] and previous FTIR data that indicate that the helical axes of lipid associated apoE are perpendicular to the axes of the fatty acyl chains of phospholipids in discoidal rHDL [ 47 , 49 ]. Further attempts were made to distinguish between anti-parallel and hairpin conformation by mixing lipid-free apoE3 (or apoE4) bearing single Cys on H2 and C1 in a 1:1 molar ratio prior to reconstitution into discoidal particles, followed by cross-linking as described.…”

“…Taken together, the decreased excimer emission coupled to lack of cross-linking suggests that intact apoE molecules are oriented in an anti-parallel, out-of-sync parallel or hairpin-loop orientation. Previous studies suggest an anti-parallel orientation of neighboring molecules in apoE3 in discoidal complexes and a hairpin loop for apoE4 [ 33 , 44 – 46 ]. Further, the hairpin model of apoE4 was proposed to adopt two possible conformations in solution, where the NT domain four-helix helix bundle was either partially open or remained intact [ 47 ].…”

Μελέτη Του Ρόλου Γονιδιακών Παραγόντων Σε Ισχαιμικά Αγγειακά Εγκεφαλικά Επεισόδια

“…Απαρτίζεται από δύο τόπους με διαφορετική λειτουργικότητα. Ένα Ν-τελικό τμήμα 22 kDa (1-191 αμινοξέα) που περιέχει την περιοχή πρόσδεσης με τον LDL υποδοχέα (136 -150 αμινοξέα) και ένα C-τελικό τμήμα 10 kDa (216 -299 αμινοξέα) υψηλής λιπιδικής συγγένειας, που ενώνει την apoE με τα λιποπρωτεϊνικά σωματίδια(7) (8). Το Ν-τελικό τμήμα περιέχει και την περιοχή πρόσδεσης με τα HSPG μόρια(12).…”